Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli.

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.

A versatile class of cell surface directional motors gives rise to gliding motility and sporulation in Myxococcus xanthus.

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.

Motor-driven intracellular transport powers bacterial gliding motility.

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.

Emergence and modular evolution of a novel motility machinery in bacteria.

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena.

Human Brain Organoids as Models for Central Nervous System Viral Infection.

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.

Functional Intercellular Transmission of miHTT via Extracellular Vesicles: An In Vitro Proof-of-Mechanism Study.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by GAG expansion in exon 1 of the huntingtin (HTT) gene. AAV5-miHTT is an adeno-associated virus serotype 5-based vector expressing an engineered HTT-targeting microRNA (miHTT). Preclinical studies demonstrate the brain-wide spread of AAV5-miHTT following a single intrastriatal injection, which is partly mediated by neuronal transport. miHTT has been previously associated with extracellular vesicles (EVs), but whether EVs mediate the intercellular transmission of miHTT remains unknown. A contactless culture system was used to evaluate the transport of miHTT, either from a donor cell line overexpressing miHTT or AAV5-miHTT transduced neurons. Transfer of miHTT to recipient (HEK-293T, HeLa, and HD patient-derived neurons) cells was observed, which significantly reduced HTT mRNA levels. miHTT was present in EV-enriched fractions isolated from culture media. Immunocytochemical and in situ hybridization experiments showed that the signal for miHTT and EV markers co-localized, confirming the transport of miHTT within EVs. In summary, we provide evidence that an engineered miRNA-miHTT-is loaded into EVs, transported across extracellular space, and taken up by neighboring cells, and importantly, that miHTT is active in recipient cells downregulating HTT expression. This represents an additional mechanism contributing to the widespread biodistribution of AAV5-miHTT.

A tool named Iris for versatile high-throughput phenotyping in microorganisms.

Advances in our ability to systematically introduce and track controlled genetic variance in microorganisms have, in the past decade, fuelled high-throughput reverse genetics approaches. When coupled to quantitative readouts, such approaches are extremely powerful at elucidating gene function and providing insights into the underlying pathways and the overall cellular network organization. Yet, until now, all efforts to quantify microbial macroscopic phenotypes have been restricted to monitoring growth in a small number of model microorganisms. We have developed an image analysis software named Iris, which allows for systematic exploration of a number of orthogonal-to-growth processes, including biofilm formation, colony morphogenesis, envelope biogenesis, sporulation and reporter activity. In addition, Iris provides more sensitive growth measurements than currently available software and is compatible with a variety of different microorganisms, as well as with endpoint or kinetic data. We used Iris to reanalyse existing chemical genomics data in Escherichia coli and to perform proof-of-principle screens on colony biofilm formation and morphogenesis of different bacterial species and the pathogenic fungus Candida albicans. We thereby recapitulated existing knowledge but also identified a plethora of additional genes and pathways involved in both processes.

Phenotype inference in an Escherichia coli strain panel.

Understanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Combining high-throughput gene function assays with mechanistic models of the impact of genetic variants is a promising alternative to genome-wide association studies. Here we have assembled a large panel of 696 Escherichia coli strains, which we have genotyped and measured their phenotypic profile across 214 growth conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across all strains. Finally, we combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to compute the growth defects of each strain. Not only could we reliably predict these defects in up to 38% of tested conditions, but we could also directly identify the causal variants that were validated through complementation assays. Our work demonstrates the power of forward predictive models and the possibility of precision genetic interventions.

An evolutionary link between capsular biogenesis and surface motility in bacteria.

Studying the evolution of macromolecular assemblies is important to improve our understanding of how complex cellular structures evolved, and to identify the functional building blocks that are involved. Recent studies suggest that the macromolecular complexes that are involved in two distinct processes in Myxococcus xanthus - surface motility and sporulation - are derived from an ancestral polysaccharide capsule assembly system. In this Opinion article, we argue that the available data suggest that the motility machinery evolved from this capsule assembly system following a gene duplication event, a change in carbohydrate polymer specificity and the acquisition of additional proteins by the motility complex, all of which are key features that distinguish the motility and sporulation systems. Furthermore, the presence of intermediates of these systems in bacterial genomes suggests a testable evolutionary model for their emergence and spread.